PROJECT SUMMARY/ABSTRACT The obesity pandemic continues unabated in Western societies and it is rising dramatically in the rest of the world. Coinciding with the rise of obesity worldwide there has been a proportional increase in highly prevalent disorders such as cardiovascular disease, type 2 diabetes and fatty liver disease. It is well established that obesity is associated with systemic chronic inflammation and that this inflammatory state contributes to the development of insulin resistance (IR); which is a hallmark in the pathogenesis of obesity-associated diseases. The elevated inflammatory status of obese individuals primarily originates from immune cells within visceral adipose tissue (VAT). Thus, understanding the molecular mechanisms that lead to VAT inflammation and IR is essential for the development of future therapies. Although studies in the last decade have uncovered key cytokines and immune cell populations that promote VAT chronic inflammation and IR, the triggering events that lead to immune cell dysfunction and VAT inflammation during obesity are poorly understood. In recent years, it has become clear that miRNAs are critical regulators of immune cells; however, little is known about how families of miRNAs regulate VAT chronic inflammatory processes during obesity. miR-181 is a highly conserved family of six miRNAs encoded by three separate clusters that is preferentially expressed in the hematopoietic system. Using miR-181-deficient mouse models, we have made three key observations that suggest that this miRNA family might play an important role in the development of VAT inflammation and IR: (i) All miR-181 family members are significantly upregulated in VAT during diet induced obesity (DIO) in mouse and humans, (ii) genetic ablation of the miR-181 family leads to significantly reduced total fat mass and improved insulin sensitivity, and (iii) the miR-181 family is critical for the function of immune cells known to infiltrate VAT during DIO. Thus, we hypothesize that the miR-181 family is expressed in specific cell types within VAT where it is instrumental in regulating gene expression programs that determine insulin sensitivity and susceptibility to obesity. Using state-of-the-art methodologies this proposal seeks to identify the cell-specific functions of the miR-181 family in VAT using a murine model of DIO. In Aim 1, we will use novel mouse models to identify the cell type within VAT in which the miR-181 family exerts its effects on insulin sensitivity and body composition. In Aim 2, we will use the CRISPR/Cas9 genome editing technology to establish the transcription factors that induce the expression of the miR-181 family within VAT during DIO. Finally, in aim 3, we will identify the targets of miR-181 within immune cells that determine insulin sensitivity in the context of obesity. In summary, this proposal will test a previously unexplored function of the miR-181 family and represents a novel avenue in obesity research. Furthermore, the information obtained during these studies could potentially lead to the identification of novel therapeutic targets for to treat obesity-associated disorders.